This invention is directed to certain peptides which have been found to cleave nucleic acid under controllable conditions. In accordance with certain preferred embodiments, members of the family of peptides, known as zinc finger peptides, have displayed the ability to cleave single stranded RNA. Diagnostic, therapeutic and research regimes are now possible featuring the cleavage function of such peptides and complexes comprising them.
Zinc finger peptides comprise a class of protein which are known per se. See: Page, D. C., Mosher, R., Simpson, E., Fisher, E. M. C., Mardon, G., Pollack, J., McGillirray, B., de la Chapelle, A., and Brown, L. G. (1987) Cell 51, 1091-1104; and Weiss, M. A., and Keutmann, H. T. (1990) Biochemistry 29, 9808-9813. The entirety of each of the references cited in this specification is incorporated herein by reference. Such peptides, which are of moderate length, are known to include zinc as a coordinate ion, and are known to complex certain nucleic acids. It has previously been know that certain zinc finger peptides complex with certain nucleic acids. However, it has now been found that at least certain of such peptides, under controllable conditions, can have functions far different than heretofore suspected. Such new functionalities have been found to give rise to the use of zinc finger peptides and related molecules in therapeutics, in diagnostics and in research.
It has now been found quite surprisingly that, under controllable conditions, zinc finger peptides can selectively cleave nucleic acids. This observation is startling in view of the fact that such peptides are known to actually complex with nucleic acids. In particular, it has been found that when the zinc concentration attendant to a zinc finger peptide decreases, the peptide tends to homodimerize, forming a structure which cleaves nucleic acid, especially single stranded RNA.
For example, a 30 amino acid peptide has been identified from among the family of zinc finger peptides, which efficiently cleaves single strand RNA. The peptide sequence corresponds to a single zinc finger of the human male associated ZFY protein; a transcription factor belonging to the Cys2His2 family of zinc finger proteins. See the Mosher, Kochoyan and Weiss references, supra. The peptide:
HOOC-KTYQCQYCEY RSADSSNLKTHIKTKHSKEK-NH2 (SEQ ID No. 1) has been shown to give rise to RNA cleavage, but such cleavage was observed only in the general absence of zinc. It has been found that coordination of the peptide with zinc resulted in complete loss of ribonuclease activity. The kinetics of RNA cleavage for the zinc-free form of the finger peptide were comparable to rates observed for the Group II intron ribozymes, e.g., V0, Kcat and Km of 6 nmol l-1 min-1, 0.96 s-1 and 105 nM, respectively). See Xiang, Q., Zhifeng Qin, P., Michels, W. J., Freeland, K., and Pyle, A. M. (1998) Biochemistry 37, 3839-3849.
The foregoing peptide appear to specifically cleave pyrimidines and preferentially cleave 5xe2x80x2-pyr-A-3xe2x80x2 within single stranded RNA. RNA sequences containing 5xe2x80x2-pyr-A-3xe2x80x2 were cleaved 10-30 fold faster than were those without such pyrimidine. The RNA cleavage products included a 3xe2x80x2-phosphate and 5xe2x80x2-hydroxyl. Single strand DNA, double strand RNA and DNA, and 2xe2x80x2-methoxy modified sequences were not hydrolyzed by this member of the class of zinc finger peptides, thus demonstrating selectivity. Divalent cations, (e.g., Mg2+ and Mn2+) were shown not to be required for catalysis. The pH for RNA cleavage with this peptide was pH 7.0. The bell shaped pH profile exhibited by the peptide is characteristic of general acid-base catalytic mechanisms observed with ribonucleases. Herries, D. G., Mathias, A. P., Rabin, B. R. (1962) Biochem. J. 85, 127; Del Rosario, E. J., Hammes, G. G. (1969) Biochemistry 8, 1884; Blackburn, P., and Moore, S. (1982) in The Enzymes (P. D. Boyer, ed.) 3rd ED. Vol. 15, pp435-468. Academic Press. N. Y.
Other zinc finger peptides, especially peptide mutants, are believed to exhibit the nucleic acid cleaving properties. Mutants to the peptide of Seq. I.D. No. 1 were synthesized in which Ala was substituted for His (H21,26A) and Glu (E9A). These mutants exhibited a 3-8 fold reduction in the cleavage rate but did not result in complete ablation in activity. This suggests a catalytic mechanism other than the one proposed for RNases A and T1. Blackburn, P., and Moore, S. (1982) in The Enzymes (P. D. Boyer, ed.) 3rd ED. Vol. 15, pp435-468. Academic Press. N.Y.; Takahashi, K and Moore, S. (1982) in The Enzymes (P. D. Boyer, ed.) 3rd ED. Vol. 15, pp435-468. Academic Press. N. Y.; and Roberts, G. C. K., Dennis, E. A., Meadows, D. H., Cohen, J. S., and Jardetzky, O. (1969) Proc. Natl. Acad. Sci. U.S.A. 62, 1151-1158.
Complete loss in ribonuclease activity was observed with Ala substitution of Lys residues (K19,20,23,25,28,30A) within the basic carboxyl terminal half of the peptide. These data suggest that positively charged basic region of the peptide may likely be binding electrostatically to the substrate. Polyacrylamide gel electrophoresis and mass spectral analysis indicated that the molecular weight of the peptide was consistent with peptide dimerization. The lack of dimer formation observed with the C5,8A peptide mutant suggest that peptide dimerization is via intermolecular disulfide bonds. Both monomer forms of the peptide, (e.g., C5,8A mutant and the reduced wild type peptide), exhibited no ribonuclease activity. Ribonuclease activity of the monomer form was regained with the substitution of Arg for Lys (K19,20,23,25,28,30R) suggesting that dimerization likely serves to enhance the binding of the peptide to the substrate but is not required for catalysis.
The ability of the zinc finger peptides to cleave nucleic acid, especially single stranded RNA, gives rise to their utility as therapeutics, diagnostics and research reagents. The destruction or disablement of RNA can be a useful event in interfering with disease states, as is well known to persons of skill in the art. Moreover, the ability to cleave single stranded RNA, especially selectively with respect to other nucleic acids and selectively with respect to situs of cleavage makes these molecules useful for research where such selective cleavage can readily be employed to advantage. Similarly, diagnosis of disease may be had through employment of zinc finger peptides to cleave RNA in a predictable fashion. These uses are particularly enhanced due to the fact that the activity is zinc dependent. Thus, by control, especially automated control, of zinc ion concentration, control of the cleavage reactions can be had. It will be appreciated that the zinc ion concentration is related to homodimer formation in the peptides. Thus, zinc ion formation and homodimer formation are related phenomena such that either or both can be used to monitor cleavage activity.
The zinc finger peptides useful in the practice of this invention are any of the family of such peptides together with mutants thereof which retain the ability to cleave nucleic acid under some controllable conditions such as zinc ion concentration. Mutants which improve specificity, reaction rate, or control functions are particularly preferred. While homodimer formation is a presently preferred interim assemblage preparatory to cleavage activity, such is not necessarily the case in some embodiments.
The ability of zinc finger peptides to selectively and controllably cleave nucleic acid, especially RNA make possible the preparation of zinc finger peptide complexes with particular activity. The covalent attachment of a zinc finger peptide to a targeting moiety, which can localize the peptide to a nucleic acid, such as an mRNA, whose cleavage or inactivation is desired, may be attained. Such attachment may be either directly, such that the peptide is directly bonded to such targeting moiety, or indirectly, whereby the peptide and targeting moiety are connected by a tether. In either case, the targeting moiety may be seen to localize the complex on or near a preselected nucleic acid, such as an mRNA important to disease. Such localization permits a high fidelity and rate of cleavage of the preselected RNA by the peptide.
Targeting moieties may be any molecule which binds specifically with a preselected nucleic acid labile to the zinc finger peptide. Oligonucleotides, such as antisense oligonucleotides are greatly preferred since they are known, are faithful in their specific binding, they can easily be synthesized and they are useful in drug, diagnostic and research formulations. Other targeting species such as small molecules, peptides, organometallics and the like may also be used.
The targeting moiety is bonded to the peptide in any convenient way such as through formation of an amide bond at the terminal carboxyl of the peptide, via a side chain function, through reaction at the terminal amine function or otherwise. Such reactions may either be directly with the targeting moiety or with a tether attached or to be attached thereto.
Tethers can be any molecule capable of bonding with both a targeting molecule or moiety and with a zinc finger peptide. Polyfunctional molecules are preferred as will be seen.
In use, zinc finger peptides of this invention are caused to contact a nucleic acid to be cleaved, especially an RNA. Preferably, the zinc concentration is reduced such that at least about 50 mole percent of the peptide is uncomplexed. Homodimer formation is believed to ensue with attendant, controlled cleavage. This mechanism may either directly cleave an RNA, such as a messenger RNA implicated in disease, thus diminishing the effects of the disease or can be used for diagnostics. Moreover, the phenomenon can be used in research where nucleic acid cleavage is widely used. The zinc ion dependence of the activity is especially useful, since the activity can, in effect, be switched on and off and, indeed, potentiated.